8-OH-DPAT (5-HT1A agonist) Attenuates 6-Hydroxy- dopamine-induced catalepsy and Modulates Inflammatory Cytokines in Rats.

Objective(s): Neuroinflammation in Parkinson disease (PD) is associated with glial cells activation and production of different inflammatory cytokines. In this study, we investigated the effect of chronic administration of 8-OH-DPAT on 6-OHDA-induced catalepsy and levels of inflammatory cytokines in cerebrospinal fluid (CSF). Materials and Methods: Catalepsy was induced by unilateral infusion of 6-OHDA (8 μg/2 μl/rat) into the central region of the sabstantia nigra pars compacta (SNc) being assessed by the bar-test, 5, 60, 120 and 180 min after intraperitoneal (IP) administration of 8-OH-DPAT (5-HT1A receptor agonist; 0.25, 0.5 and 1mg/kg, IP for 10 days). CSF samples were collected on the tenth day of 8-OH-DPAT administration and analyzed by ELISA method to measure levels of TNF-α, IL-1β and IL-6. Results: Chronic injection of 8-OH-DPAT decreased catalepsy in a dose dependent manner when compared with the control group. The most anti-cataleptic effect was observed at the dose of 1 mg/kg of 8-OH-DPAT. Levels of TNF-α in CSF increased three weeks after 6-OHDA injection while there was a significant decrease in TNF-α level of parkinsonian animals treated with 8-OH-DPAT (1 mg/kg, IP for 10 days). IL-1β and IL-6 decreased and increased in parkinsonian rats and in 8-OH-DPAT-treated parkinsonian rats, respectively. Conclusion: Our study indicated that chronic administration of 8-OH-DPAT improves catalepsy in 6-OHDA-induced animal model of PD and restores central concentration of inflammatory cytokines to the basal levels. 5-HT1A receptor agonists can be suggested as potential adjuvant therapy in PD by modulation of cerebral inflammatory cytokines.


Introduction
Parkinson diseas e (PD) is the second most common and progressive neurodegenerative disease caused mainly by loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). The major movement symptoms are ri gidity, akinesia, tremor and postural abnormalities as well as cogni tive disturbances (1).
The role of neuroinfl ammation in degeneration of nigrostriatal neurons is of interest to many investigators (2,3). The first evidence for the rol e of inflammation in PD came from an observation by McGeer and colleagues on activated microglia and T cells in the pos t-mortem SNc of a patient suffering from PD (4). Epidemiological studies hav e shown that the incidence of idiopathic PD is lower in chronic users of anti-inflammatory drugs (5,6). Neuroinflammation is regulated by many signal molecules including cytokines. They are multifuncti-onal proteins and in the CNS, pl ay a role in the normal dev elopment of the brain as well as in neuroimmuno-pathological processes following injury and neurodegeneration (7). Several studies have reported significant increase of pro -inflammatory cytokines such as IFN-γ, IL-1β and TNF-α, being expressed by glial cells in the nigrostratial regions of pati ents with PD (4,(8)(9)(10). In general, proinflammatory cytoki nes such as TNF -α has neurotoxic effects, while IL -6 and IL-1β, classical proinflammatory cytoki nes, have a dual effect. For instance, low concentrations of IL -6 protect neuronal cells from death, while larger concentrations are neurotoxic (11,12).

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in the regulation of movements executed by the basal ganglia (14). In this context, role of 5-HT1A receptors in motor impairments of PD is the center for attention (13,16). Activation of these receptors could decreas e serotonin release and subsequently improve motor function in 6-OHDA-lesioned rats (17,18). Studies have shown that s timulation of the 5-HT1A receptor attenuates anoxia-induced apoptosis in the neuronal HN2-5 (Hippocampal neuronderived cell line) cells (19). It seems that cytokines act as messengers between the immune system and the brain, exerting their effect on sero tonergic system through several processes such as degradation of their precursor, tryptophan (20). The role of pro-inflammatory cytokines in the pathogenesis of PD has been shown in s everal studies (12,21,22), although the effect of chronic administration of 5-HT1A receptor agonists on 6-OHDA-induced catalepsy and the rol e of cytokines such as TNF-α, IL-1β and IL-6 has not been clearly studied yet. Thus, in this study we attempted to investigate the effect of chronic administration of 8-OH-DPAT on 6-OHDA-induced catalepsy and possible involvement of TNF-α, IL-1β and IL-6.

Chemicals
All chemicals were obtained from Sigma Chemical Co. (USA), except for ELISA kits, which were purchas ed from eBioscience Co. (Austri a). All solutions were prepared freshly on the experimentation day. 8-OH-DPAT (a 5-HT1A receptor agonist) and 6-OHDA were dissolved in physiological saline (0.9% NaCl) and 0.9% saline containing 0. 2% (w/v) ascorbic acid, respectively. 6-OHDA was injected into the central region of the substantia nigra pars compacta (SNc) in a total volume of 2 μl /rat wi th a constant i njection rate of 0.2 μl /min.

Animals
The experiments were carried out on male Wistar rats wei ghing 270-300 g. Animals were housed in standard polypropylene cages, four per cage, under a 12:12 hr light/dark schedule at an ambient temperature of 25 ± 2°C and were allowed food and water ad libitum. Animals were acclimated to the testi ng conditions for 2 days before the behavioral experiment was conducted. All procedures were carried out under the ethical guidelines of the Tabriz University of Medical Sciences. Twenty-four rats were divided into three groups: normal, sham-operated (receiving 2 μl vehicle) and 6-OHDA (8 μg/2 μl/rat)-injected.

6-OHDA-induced SNc lesion
Animals were anesthetized wi th an IP injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). After being deeply anesthetized (loss of corneal and toe pad reflexes), rats were mounted in a Stoelting stereotaxic frame in the flat skull position. The scalp was shaved, swabbed with povidone-iodine 10%, and a central incision made to expose the skull. 6-OHDA was i njected thorough a gui de cannula (23 gauge stainless steel) implanted in the SNc. The coordinates for this site were based on the rat brain atlas (23) as follows: anteroposterior (AP): -5.0 mm from the bregma; mediolateral (ML): -2.1 mm from the midline and dorsoventral (DV): -7.7 from the skull.
Desipramine (25 mg/kg, IP) was injected 30 min before the intra-SNc injection of 6-OHDA to avoid destruction of noradrenergic neurons. Thereafter, 6-OHDA (8 μg per rat in 2 μl saline with 0.2% ascorbic acid) was infused with an infusion pump at a constant flow rate of 0.2 μl/min into the l eft SNc. At the end of the infusion, the injection tube was kept implanted for an additional 2 min and then was slowly retracted. Sham -operated animals were submitted to the s ame procedure but 2 μl vehicle (0.9% saline contai ning 0.2% (w/v) ascorbic acid) instead of 6-OHDA was infused into the SNc.

Catalepsy test
Catalepsy was measured using a standard bar test 21 days after 6-OHD A and 10 days after IP injection of 8-OH-D PAT. In this method, forepaws of rats were pl aced ov er a 9-cm-high standard wooden bar, and the duration of retention of rats in this imposed posture was considered as the bar test elapsed time. The end poi nt of catalepsy was considered when both front paws were removed from the bar or when the animal moved its head in an exploratory manner. The cut-off time of the test was 600 sec. The tes t was carried out 5, 60, 120 and 180 mi n after drug administration on the 10 th day.
All observations were made between 9 am and 4 pm. After a three-week recovery period, only the rats being markedly immobilized in the bar test were subjected to further experimentation (parki nsonian rats). Afterwards, the parkinsonian rats were randomly divided into equal groups and received IP injections of 8-OHD-PAT (0.25, 0.5 and1 mg/kg, IP) once daily (9 a.m.) for 10 days.

CSF sampling
CSF samples were collected on day 10 of 8-OH-DPAT administration. Animals were anesthetized by IP i njections of ketamine (50 mg/kg) and xylazine (5 mg/kg) and mounted in a Stoelti ng stereotaxic frame. The skull was kept i n 45 ° position and CSF was aspirated using a sterile 100 µl syringe 23gauge needle. The CSF s amples were kept at -70°C until being assessed by Enzyme-linked immunosorbent assays (ELISA) method.

Analysis of TNF-α, IL-1β an d IL-6 expression by ELISA
ELISA method was employed for determination of TNF-α, IL-1β and IL-6 i n CSF samples. Assays were Briefly, the frozen CSF s amples were diluted, added into the wells and i ncubated at room temperature for 120 min on a microplate shaker. Subsequent to washing, diluted Streptavidin-Hors eradish peroxidas e-conjugated antimouse TNF A, IL -1B and IL-6 were reacted for 60 min at room temperature (on microplate shaker set at 200 rpm). After washing for s econd time, the wells were developed with tetramethyl benzidi ne (TMB) for 10 min and the optical densities were read at 450 nm with an ELISA reader.

Histology
All animals having guide cannula were sacrificed at the end of the experiments. Brain dissections were performed in all animals to confirm the exact implantation of guide cannula into the SNc. Brain in the injecti ng tube in situ was fixed in 10% formalin for 1 week. The location of the tip of the injecting tube was then verified i n seri al sections. Only the results from bar tests in animals with the tip of the injecting tube withi n the SNc area were us ed for statistical analysis.

Statistical analysis
Statistical analysis for each data set was calculated by SPSS software (version 16.0). Data were expressed as mean+SEM, and one-way ANOVA test was utilized to analyze in the data from behavioral and biochemical experiments. In the case of significant v ariation (P< 0.05), the values were compared by Tukey test.

Effect of 8-OH-DPAT on inflammatory parameters
Results showed that TNF -α was undetectable in normal rats while it was increas ed three weeks after 6-OHDA injection and decreased following 10 days of IP administeration of 8-OH-DPAT to normal animals. Levels of the two other cytokines were decreased in 6-OHDA-lesioned rats in comparison wi th normal group but increased 10 days after i njection of 8-OH-DPAT ( Figure 3).

Discussion
In our previous studies (13,24) the potential anticataleptic effects of 5-HT1A receptor agonists in 6-OHDA-lesioned rats has been inv estigated i n single dose administrations. Given the chronic clinical administrations of such drugs, we investigated the potenti al anticataleptic effects of chronic administration of 8-OH-D PAT in 6-OHDA-l esioned rats in the current study. Our results showed that intra-SNc injection of 6-OHDA induced catalepsy in animals when assessed by the bar tes t (13). Bar test is a standard test being frequently used for evaluation of catalepsy induced by 6-OHDA and neuroleptic drugs in rodents (25). According to the results, chronic admi nistration of 8-OH-D PAT, an agonist of 5-HT1A receptors, improved catalepsy in 6-OHDA-lesioned rats in a dose dependent manner. Such fi nding confi rms the previous studi es, reporting a promising role for 5-HT1A agonists in decreasing the motor disorders associated with PD (24). 5-HT1A receptors are widely distributed throughout the bas al ganglia. They are located on dorsal raphe neurons with efferents to the stri atum and on cortical neurons that s end glutamatergic projections into the basal ganglia (26). In the basal ganglia, serotonin modulates dopamine-related motor activity through affecting the 5-HT1A receptor (27). It has been shown that 5-HT1A agonists improve motor impai rments in parkinsonian animals via stimulation of somatodendritic 5-HT1A receptors and subsequent decrease in serotonin release from the nerv e endings (28). Furthermore, studies have indicated that 5-HT1A receptor plays a role in neuronal survival (29,30) and has a neuroprotective effect in animal models of stroke and traumatic brain injury (29,31). Such effect is exerted through inhibition of glutamate release that leads to a reduction in the putative excitotoxicity-mediated cell death (31).
Herein, we assessed levels of inflammatory cytokines i.e TNF-α, IL-6 and IL-1β in the CSF of parki nsonian rats being treated with chronic injections of 8-OH-DPAT. According to our results, there was a significant increas e in the amount of TNF-α in parkinsonian rats whereas its levels were resorted to normal ranges by chronic administration of 8-OH-D PAT. This is in accordance with other studies reporting that toxic effects of 6 -OHDA are in part mediated through the activation of microglia and increasing levels of TNF-α in both SN and striatum (4,9,32 ). The substantia nigra (SN) has high density of microglia and it is hy pothesized that DA neurons are susceptible to inflammatory damage as a major stimuli for neurodegenerative diseases (33). Activated microglia rel ease proinflammatory cytokines such as TNF -α that play a key role in modulation of inflammatory respons es (34).
The levels of IL-1β and IL-6 were decreased in parki nsonian rats when compared with normal (nonparki nsonian) animals. In parkinsonian rats, which were treated with chronic injections of 8-OH-D PAT, the levels of IL-1β and IL-6 were increas ed to that of normal rats. It has been reported that there is an increase in CSF concentration of IL -1β and IL-6 in parki nsonian rats (32,35). In addi tion to their proinflammatory effect, thes e are pleiotropic cytoki nes, which can produce neuroprotective effects in PD, Alzheimer disease (AD) and CNS injuri es (32,36). Our results showed that IL -1β and IL-6 were decreas ed in 6-OHDA-lesioned rats while in 6-OHDAlesioned rats, bei ng treated wi th 8-OH-D PAT, the levels of IL-1β and IL-6 were res tored to normal values. This is in agreement with previous studies which sugges t a neuroprotective effect for these cytokines (22,37).

Conclusion
Our data suggest that chronic administration of 8-OH-D PAT improv es catalepsy in 6-OHDA-l esioned rats. Moreover, we suggest that 5-HT1A receptor agonists can be utilized as adjuvant therapy along with commonly used anti-parkinsonian drugs. However, further clinical investigations should be carri ed out to prov e this.